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. 1994 Jun;14(6):3559–3568. doi: 10.1128/mcb.14.6.3559

Dorsal, a Drosophila Rel-like protein, is phosphorylated upon activation of the transmembrane protein Toll.

S K Gillespie 1, S A Wasserman 1
PMCID: PMC358723  PMID: 8196601

Abstract

The nuclear import of dorsal, a Drosophila Rel homolog, is directed by a spatially restricted extracellular ligand in blastoderm embryos. We have demonstrated both that dorsal is an embryonic phosphoprotein and that its phosphorylation state is regulated by an intracellular signaling pathway initiated by the transmembrane receptor Toll. Immunoblot analysis of cytoplasm from precisely staged embryos revealed that the phosphorylation state of dorsal is altered during the time period that Toll is activated. Moreover, mutations that constitutively activate Toll stimulated dorsal phosphorylation, while mutations that block Toll activation reduced the level of dorsal phosphorylation. We further demonstrated that signal-dependent dorsal phosphorylation is modulated by three intracellular proteins, pelle, tube, and cactus. Using double-mutant embryos, we then explored the nature of the kinase activity responsible for dorsal phosphorylation. We found that free dorsal is a substrate for a signal-independent kinase activity. In addition, our results imply that dorsal is a substrate for a Toll-dependent kinase. These results are consistent with the hypothesis that phosphorylation of Rel-related proteins may be required for the proper nuclear localization and transcriptional activity of these proteins.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson K. V., Jürgens G., Nüsslein-Volhard C. Establishment of dorsal-ventral polarity in the Drosophila embryo: genetic studies on the role of the Toll gene product. Cell. 1985 Oct;42(3):779–789. doi: 10.1016/0092-8674(85)90274-0. [DOI] [PubMed] [Google Scholar]
  2. Anderson K. V., Nüsslein-Volhard C. Information for the dorsal--ventral pattern of the Drosophila embryo is stored as maternal mRNA. Nature. 1984 Sep 20;311(5983):223–227. doi: 10.1038/311223a0. [DOI] [PubMed] [Google Scholar]
  3. Baeuerle P. A., Baltimore D. Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-kappa B transcription factor. Cell. 1988 Apr 22;53(2):211–217. doi: 10.1016/0092-8674(88)90382-0. [DOI] [PubMed] [Google Scholar]
  4. Baeuerle P. A., Baltimore D. I kappa B: a specific inhibitor of the NF-kappa B transcription factor. Science. 1988 Oct 28;242(4878):540–546. doi: 10.1126/science.3140380. [DOI] [PubMed] [Google Scholar]
  5. Baeuerle P. A. The inducible transcription activator NF-kappa B: regulation by distinct protein subunits. Biochim Biophys Acta. 1991 Apr 16;1072(1):63–80. doi: 10.1016/0304-419x(91)90007-8. [DOI] [PubMed] [Google Scholar]
  6. Beg A. A., Finco T. S., Nantermet P. V., Baldwin A. S., Jr Tumor necrosis factor and interleukin-1 lead to phosphorylation and loss of I kappa B alpha: a mechanism for NF-kappa B activation. Mol Cell Biol. 1993 Jun;13(6):3301–3310. doi: 10.1128/mcb.13.6.3301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Beg A. A., Ruben S. M., Scheinman R. I., Haskill S., Rosen C. A., Baldwin A. S., Jr I kappa B interacts with the nuclear localization sequences of the subunits of NF-kappa B: a mechanism for cytoplasmic retention. Genes Dev. 1992 Oct;6(10):1899–1913. doi: 10.1101/gad.6.10.1899. [DOI] [PubMed] [Google Scholar]
  8. Bird T. A., Sleath P. R., deRoos P. C., Dower S. K., Virca G. D. Interleukin-1 represents a new modality for the activation of extracellular signal-regulated kinases/microtubule-associated protein-2 kinases. J Biol Chem. 1991 Nov 25;266(33):22661–22670. [PubMed] [Google Scholar]
  9. Blank V., Kourilsky P., Israël A. NF-kappa B and related proteins: Rel/dorsal homologies meet ankyrin-like repeats. Trends Biochem Sci. 1992 Apr;17(4):135–140. doi: 10.1016/0968-0004(92)90321-y. [DOI] [PubMed] [Google Scholar]
  10. Bomsztyk K., Rooney J. W., Iwasaki T., Rachie N. A., Dower S. K., Sibley C. H. Evidence that interleukin-1 and phorbol esters activate NF-kappa B by different pathways: role of protein kinase C. Cell Regul. 1991 Apr;2(4):329–335. doi: 10.1091/mbc.2.4.329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Bomsztyk K., Toivola B., Emery D. W., Rooney J. W., Dower S. K., Rachie N. A., Sibley C. H. Role of cAMP in interleukin-1-induced kappa light chain gene expression in murine B cell line. J Biol Chem. 1990 Jun 5;265(16):9413–9417. [PubMed] [Google Scholar]
  12. Brautigan D. L., Shriner C. L. Methods to distinguish various types of protein phosphatase activity. Methods Enzymol. 1988;159:339–346. doi: 10.1016/0076-6879(88)59034-1. [DOI] [PubMed] [Google Scholar]
  13. Chasan R., Jin Y., Anderson K. V. Activation of the easter zymogen is regulated by five other genes to define dorsal-ventral polarity in the Drosophila embryo. Development. 1992 Jun;115(2):607–616. doi: 10.1242/dev.115.2.607. [DOI] [PubMed] [Google Scholar]
  14. Cordle S. R., Donald R., Read M. A., Hawiger J. Lipopolysaccharide induces phosphorylation of MAD3 and activation of c-Rel and related NF-kappa B proteins in human monocytic THP-1 cells. J Biol Chem. 1993 Jun 5;268(16):11803–11810. [PubMed] [Google Scholar]
  15. Gay N. J., Keith F. J. Drosophila Toll and IL-1 receptor. Nature. 1991 May 30;351(6325):355–356. doi: 10.1038/351355b0. [DOI] [PubMed] [Google Scholar]
  16. Geisler R., Bergmann A., Hiromi Y., Nüsslein-Volhard C. cactus, a gene involved in dorsoventral pattern formation of Drosophila, is related to the I kappa B gene family of vertebrates. Cell. 1992 Nov 13;71(4):613–621. doi: 10.1016/0092-8674(92)90595-4. [DOI] [PubMed] [Google Scholar]
  17. Ghosh S., Baltimore D. Activation in vitro of NF-kappa B by phosphorylation of its inhibitor I kappa B. Nature. 1990 Apr 12;344(6267):678–682. doi: 10.1038/344678a0. [DOI] [PubMed] [Google Scholar]
  18. Gillespie P. G., Hudspeth A. J. Chemiluminescence detection of proteins from single cells. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2563–2567. doi: 10.1073/pnas.88.6.2563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gillespie P. G., Hudspeth A. J. High-purity isolation of bullfrog hair bundles and subcellular and topological localization of constituent proteins. J Cell Biol. 1991 Feb;112(4):625–640. doi: 10.1083/jcb.112.4.625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gordon J. A. Use of vanadate as protein-phosphotyrosine phosphatase inhibitor. Methods Enzymol. 1991;201:477–482. doi: 10.1016/0076-6879(91)01043-2. [DOI] [PubMed] [Google Scholar]
  21. Govind S., Steward R. Dorsoventral pattern formation in Drosophila: signal transduction and nuclear targeting. Trends Genet. 1991 Apr;7(4):119–125. doi: 10.1016/0168-9525(91)90456-z. [DOI] [PubMed] [Google Scholar]
  22. Hashimoto C., Gerttula S., Anderson K. V. Plasma membrane localization of the Toll protein in the syncytial Drosophila embryo: importance of transmembrane signaling for dorsal-ventral pattern formation. Development. 1991 Apr;111(4):1021–1028. doi: 10.1242/dev.111.4.1021. [DOI] [PubMed] [Google Scholar]
  23. Hashimoto C., Hudson K. L., Anderson K. V. The Toll gene of Drosophila, required for dorsal-ventral embryonic polarity, appears to encode a transmembrane protein. Cell. 1988 Jan 29;52(2):269–279. doi: 10.1016/0092-8674(88)90516-8. [DOI] [PubMed] [Google Scholar]
  24. Hecht P. M., Anderson K. V. Extracellular proteases and embryonic pattern formation. Trends Cell Biol. 1992 Jul;2(7):197–202. doi: 10.1016/0962-8924(92)90246-j. [DOI] [PubMed] [Google Scholar]
  25. Hecht P. M., Anderson K. V. Genetic characterization of tube and pelle, genes required for signaling between Toll and dorsal in the specification of the dorsal-ventral pattern of the Drosophila embryo. Genetics. 1993 Oct;135(2):405–417. doi: 10.1093/genetics/135.2.405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Higgins R. C., Dahmus M. E. Rapid visualization of protein bands in preparative SDS-polyacrylamide gels. Anal Biochem. 1979 Mar;93(2):257–260. doi: 10.1016/s0003-2697(79)80148-7. [DOI] [PubMed] [Google Scholar]
  27. Isoda K., Roth S., Nüsslein-Volhard C. The functional domains of the Drosophila morphogen dorsal: evidence from the analysis of mutants. Genes Dev. 1992 Apr;6(4):619–630. doi: 10.1101/gad.6.4.619. [DOI] [PubMed] [Google Scholar]
  28. Kidd S. Characterization of the Drosophila cactus locus and analysis of interactions between cactus and dorsal proteins. Cell. 1992 Nov 13;71(4):623–635. doi: 10.1016/0092-8674(92)90596-5. [DOI] [PubMed] [Google Scholar]
  29. Letsou A., Alexander S., Orth K., Wasserman S. A. Genetic and molecular characterization of tube, a Drosophila gene maternally required for embryonic dorsoventral polarity. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):810–814. doi: 10.1073/pnas.88.3.810. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lowenthal J. W., Ballard D. W., Böhnlein E., Greene W. C. Tumor necrosis factor alpha induces proteins that bind specifically to kappa B-like enhancer elements and regulate interleukin 2 receptor alpha-chain gene expression in primary human T lymphocytes. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2331–2335. doi: 10.1073/pnas.86.7.2331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. MacKintosh C., Beattie K. A., Klumpp S., Cohen P., Codd G. A. Cyanobacterial microcystin-LR is a potent and specific inhibitor of protein phosphatases 1 and 2A from both mammals and higher plants. FEBS Lett. 1990 May 21;264(2):187–192. doi: 10.1016/0014-5793(90)80245-e. [DOI] [PubMed] [Google Scholar]
  32. Norris J. L., Manley J. L. Selective nuclear transport of the Drosophila morphogen dorsal can be established by a signaling pathway involving the transmembrane protein Toll and protein kinase A. Genes Dev. 1992 Sep;6(9):1654–1667. doi: 10.1101/gad.6.9.1654. [DOI] [PubMed] [Google Scholar]
  33. Osborn L., Kunkel S., Nabel G. J. Tumor necrosis factor alpha and interleukin 1 stimulate the human immunodeficiency virus enhancer by activation of the nuclear factor kappa B. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2336–2340. doi: 10.1073/pnas.86.7.2336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Pearson R. B., Kemp B. E. Protein kinase phosphorylation site sequences and consensus specificity motifs: tabulations. Methods Enzymol. 1991;200:62–81. doi: 10.1016/0076-6879(91)00127-i. [DOI] [PubMed] [Google Scholar]
  35. Rio D. C., Laski F. A., Rubin G. M. Identification and immunochemical analysis of biologically active Drosophila P element transposase. Cell. 1986 Jan 17;44(1):21–32. doi: 10.1016/0092-8674(86)90481-2. [DOI] [PubMed] [Google Scholar]
  36. Roth S., Hiromi Y., Godt D., Nüsslein-Volhard C. cactus, a maternal gene required for proper formation of the dorsoventral morphogen gradient in Drosophila embryos. Development. 1991 Jun;112(2):371–388. doi: 10.1242/dev.112.2.371. [DOI] [PubMed] [Google Scholar]
  37. Roth S., Stein D., Nüsslein-Volhard C. A gradient of nuclear localization of the dorsal protein determines dorsoventral pattern in the Drosophila embryo. Cell. 1989 Dec 22;59(6):1189–1202. doi: 10.1016/0092-8674(89)90774-5. [DOI] [PubMed] [Google Scholar]
  38. Rushlow C. A., Han K., Manley J. L., Levine M. The graded distribution of the dorsal morphogen is initiated by selective nuclear transport in Drosophila. Cell. 1989 Dec 22;59(6):1165–1177. doi: 10.1016/0092-8674(89)90772-1. [DOI] [PubMed] [Google Scholar]
  39. Rushlow C., Warrior R. The rel family of proteins. Bioessays. 1992 Feb;14(2):89–95. doi: 10.1002/bies.950140204. [DOI] [PubMed] [Google Scholar]
  40. Schneider D. S., Hudson K. L., Lin T. Y., Anderson K. V. Dominant and recessive mutations define functional domains of Toll, a transmembrane protein required for dorsal-ventral polarity in the Drosophila embryo. Genes Dev. 1991 May;5(5):797–807. doi: 10.1101/gad.5.5.797. [DOI] [PubMed] [Google Scholar]
  41. Schüpbach T., Wieschaus E. Female sterile mutations on the second chromosome of Drosophila melanogaster. I. Maternal effect mutations. Genetics. 1989 Jan;121(1):101–117. doi: 10.1093/genetics/121.1.101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sen R., Baltimore D. Inducibility of kappa immunoglobulin enhancer-binding protein Nf-kappa B by a posttranslational mechanism. Cell. 1986 Dec 26;47(6):921–928. doi: 10.1016/0092-8674(86)90807-x. [DOI] [PubMed] [Google Scholar]
  43. Shelton C. A., Wasserman S. A. pelle encodes a protein kinase required to establish dorsoventral polarity in the Drosophila embryo. Cell. 1993 Feb 26;72(4):515–525. doi: 10.1016/0092-8674(93)90071-w. [DOI] [PubMed] [Google Scholar]
  44. Shirakawa F., Mizel S. B. In vitro activation and nuclear translocation of NF-kappa B catalyzed by cyclic AMP-dependent protein kinase and protein kinase C. Mol Cell Biol. 1989 Jun;9(6):2424–2430. doi: 10.1128/mcb.9.6.2424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Shirakawa F., Yamashita U., Chedid M., Mizel S. B. Cyclic AMP--an intracellular second messenger for interleukin 1. Proc Natl Acad Sci U S A. 1988 Nov;85(21):8201–8205. doi: 10.1073/pnas.85.21.8201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. St Johnston D., Nüsslein-Volhard C. The origin of pattern and polarity in the Drosophila embryo. Cell. 1992 Jan 24;68(2):201–219. doi: 10.1016/0092-8674(92)90466-p. [DOI] [PubMed] [Google Scholar]
  47. Stein D., Nüsslein-Volhard C. Multiple extracellular activities in Drosophila egg perivitelline fluid are required for establishment of embryonic dorsal-ventral polarity. Cell. 1992 Feb 7;68(3):429–440. doi: 10.1016/0092-8674(92)90181-b. [DOI] [PubMed] [Google Scholar]
  48. Stein D., Roth S., Vogelsang E., Nüsslein-Volhard C. The polarity of the dorsoventral axis in the Drosophila embryo is defined by an extracellular signal. Cell. 1991 May 31;65(5):725–735. doi: 10.1016/0092-8674(91)90381-8. [DOI] [PubMed] [Google Scholar]
  49. Steward R. Dorsal, an embryonic polarity gene in Drosophila, is homologous to the vertebrate proto-oncogene, c-rel. Science. 1987 Oct 30;238(4827):692–694. doi: 10.1126/science.3118464. [DOI] [PubMed] [Google Scholar]
  50. Steward R. Relocalization of the dorsal protein from the cytoplasm to the nucleus correlates with its function. Cell. 1989 Dec 22;59(6):1179–1188. doi: 10.1016/0092-8674(89)90773-3. [DOI] [PubMed] [Google Scholar]
  51. Wasserman S. A. A conserved signal transduction pathway regulating the activity of the rel-like proteins dorsal and NF-kappa B. Mol Biol Cell. 1993 Aug;4(8):767–771. doi: 10.1091/mbc.4.8.767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Whalen A. M., Steward R. Dissociation of the dorsal-cactus complex and phosphorylation of the dorsal protein correlate with the nuclear localization of dorsal. J Cell Biol. 1993 Nov;123(3):523–534. doi: 10.1083/jcb.123.3.523. [DOI] [PMC free article] [PubMed] [Google Scholar]

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